PT2908626T - Humanized il-15 animals - Google Patents

Description

"Animals with il-15 humanized"

FIELD

Non-human animals comprising, in their germ line, a humanized endogenous non-human IL-15 locus. Non-human animals comprising, in their germ line, a humanized IL-15 coding sequence under the control of endogenous non-human regulatory elements. Non-human animals (e.g., mammals, for example, rodents such as mice, rats and hamsters) comprising a genetic modification comprising a substitution, and an endogenous locus, of a non-human IL-15 gene sequence for a gene sequence of human or humanized IL-15. Rodents and other non-human animals expressing human or humanized IL-15 from a modified endogenous non-human IL-15 locus. Non-human animals expressing human or humanized IL-15 under the control of a promoter and / or non-human IL-15 regulatory sequences.

FUNDAMENTALS

Transgenic mice with randomly inserted transgenes containing a human IL-15 sequence are known in the art. However, transgenic mice expressing human IL-15 from randomly integrated transgenes are not ideal in either aspect. For example, most mice transgenic for human IL-15 exhibit abnormal levels and / or ratios of certain cells, including lymphocytes (e.g., T cells), which are probably due to dysregulation of immune cell function. These mice also exhibit a myriad of pathologies, presumably ultimately due to the deregulation of transgenic IL-15. Such deregulation may result, for example, from the absence of endogenous control elements, and / or the placement of the human IL-15 sequence away from the endogenous IL-15 locus.

There remains a need in the art of non-human animals comprising sequences encoding human IL-15, wherein the human IL-15 coding sequences are at an endogenous non-human IL-15 locus, and / or are under the regulatory elements endogenous non-human IL-15 (e.g., upstream and / or downstream non-coding regions). There is a need in the art of non-human animals which express human IL-15 under the control of endogenous non-human regulatory elements. There is a need in the art of non-human animals that express human IL-15 in a way that is physiologically relevant in the non-human animal. There is a need in the art for non-human animals which express a human IL-15, wherein non-human animals lack a significant abnormality in lymphocyte populations, for example, T cell populations. There is also a need in the art for non-human animals. human cells expressing human or humanized IL-15, and the lack of one or more of the pathologies exhibited by non-human animals that are transgenic for human IL-15. Ohta et al. (J. Immunol, 169: pp 460-468 (2002) describes Th1-type CD8 + NK1.1 + T cells resistant to cell death induced by IL-15-dependent activation for the development of small intestinal inflammation. Marks-Konczalik et al (PNAS, 97: pp 11445-11450 (2000)) discloses that IL-2 induced activation-induced cell death is inhibited in mice transgenic for IL-15. IL-5, Fehniger et al (Blood Cells, Molecules and Diseases, 27: pp 223-230 (2001)) describes fatal leukemia in transgenic mice for interleukin-15. IL-6 (Devoy et al., Nat. Rev. Genetics, 13: pp. 14-20 (2012)) describes genomically humanized mice, Steel et al (Trends in Pharmacological Sciences, 33: pp 35-41 (2011)) describes the biology of IL-15 and therapeutic implications in cancer.

SUMMARY The invention provides a genetically modified mouse whose genome comprises a substitution at a mouse endogenous IL-15 locus of a mouse genomic fragment encoding a mature mouse IL-15 polypeptide by a human genomic segment encoding a human IL-15 polypeptide, wherein the human IL-15 exons in said human genomic segments consist of exons 3, 4, 5 and 6 encoding the human IL-15 protein to form a humanized IL-15 gene in that the exons of IL-15 are numbered with the first exon coding as exon 1, and wherein the humanized IL-15 gene is under the control of all regulatory elements upstream of the endogenous mouse IL-15 at the IL locus Endogenous mouse. The invention further provides a method of producing a genetically modified mouse which comprises modifying the genome of a mouse by replacing a mouse genomic fragment encoding a mature mouse IL-15 polypeptide at the endogenous IL-15 locus by a human genomic segment encoding a mature human IL-15 polypeptide, wherein the human IL-15 exons in said human genomic segment consist of exons 3, 4, 5 and 6 encoding the human IL-15 protein to form a humanized IL-15 gene, wherein the exons of IL-15 are numbered with the first exon coding as exon 1, and wherein the humanized IL-15 gene is under the control of all upstream regulatory elements endogenous mouse IL-15 at the mouse endogenous IL-15 locus. The invention further provides a genetically modified mouse whose genome comprises a humanized IL-15 gene, wherein said humanized IL-15 gene comprises exons 1 and 2 encoding the mouse IL-15 protein and a human genomic segment, wherein exons of human IL-15 in said human genomic segment consist of exons 3, 4, 5 and 6 encoding the human IL-15 protein, wherein IL-15 exons are numbered with the first exon coding as exon 1 , and wherein the humanized IL-15 gene is under the control of all regulatory elements upstream from the mouse IL-15 of a mouse endogenous IL-15 locus, and said humanized IL-15 gene encodes a which protein comprises a mature human IL-15 polypeptide sequence. The invention also provides a method of producing a genetically modified mouse comprising introducing into the genome of a mouse a humanized IL-15 gene comprising the exons 1 and 2 encoding the mouse IL-15 protein and a segment wherein the exons of human IL-15 in said human genomic segment consist of exons 3, 4, 5 and 6 encoding the human IL-15 protein, wherein the exons of IL-15 are numbered with the first coding exon as exon 1, and wherein the humanized IL-15 gene is under the control of all regulatory elements upstream of the mouse IL-15 of a locus of the endogenous mouse IL-15, and said IL-15 gene encodes a protein comprising a mature human IL-15 polypeptide sequence. Genetically modified non-human organisms comprising a humanized IL-15 locus are described. Non-human organisms comprising a humanized IL-15 gene, wherein the humanized IL-15 gene is under the control of one or more non-human endogenous regulatory elements, are described. Non-human organisms comprising a humanized IL-15 gene are described in a non-human endogenous IL-15 locus. Non-human organisms are described which comprise a humanized endogenous IL-15 locus that is capable of being passed through the germ line of organisms. Non-human animals, for example, mammals (e.g., rodents, e.g., mice or mice) expressing human IL-15 from a modified endogenous IL-15 locus, wherein the expressed IL-15 is totally or partially human. Genetically modified non-human animals, embryos, cells, tissues and nucleic acids are described which comprise a human IL-15 genomic sequence regulated by non-human IL-15 regulatory control. Non-human animals express a humanized IL-15 protein, or fully human IL-15 protein (e.g., a fully human mature IL-15 protein), and do not exhibit one or more of the disorders of non-human animals with human IL-15 known in the art. Non-animal animals may be mammals, for example, rodents, e.g., mice, rats, hamsters, etc. The mammal may be a rodent; the rodent can be a mouse or a mouse. Genetically modified non-human animals, embryos, cells, tissues and nucleic acids comprising a human IL-15 genomic sequence at a non-human IL-15 locus are described. Non-human animals express a humanized IL-15 protein, or fully human IL-15 protein (e.g., a fully human mature IL-15 protein), a modified endogenous non-human locus regulated by one or more endogenous non-human regulatory sequences of the modified endogenous IL-15 locus, and do not exhibit one or more of the non-human animal pathologies with transgenic human IL-15 known in the art. Non-animal animals may be mammals, for example, rodents, e.g., mice, rats, hamsters, etc. The mammal may be a rodent; the rodent can be a mouse or a mouse.

Non-human animals may comprise a modified IL-15 locus in the genome of the non-human animal such that the modified IL-15 locus is capable of being passed through the germ line, wherein the modified endogenous IL-15 locus comprises a humanization of at least a mature protein coding portion of the endogenous IL-15 locus. Nonhuman animals may be mammals. Mammals may be rodents, and rodents are heterozygous or homozygous relative to the modified IL-15 locus. Rodents can be selected from mice and rats. Mice and rats may be homozygous for the modified IL-15 locus, and are unable to express an endogenous fully or completely rat IL-15 protein, and the mouse and rat express a mature human IL-15 protein. A non-human animal comprising a first wild-type endogenous IL-15 allele, and a humanization of a second endogenous IL-15 allele is described. A non-human animal is described which comprises the lack of a first allele of the endogenous IL-15 and a humanization of a second allele of the endogenous IL-15. A non-human animal comprising the lack of an endogenous functional IL-15 allele is described, and comprises at least one copy of a humanized IL-15 allele under the control of endogenous non-human regulatory elements. At least one copy of a humanized IL-15 allele may be at an endogenous IL-15 locus. Humanization may be one or more exons and / or introns at the non-human endogenous IL-15 locus. Non-human animals having a modified IL-15 locus in which an endogenous non-human 5 'untranslated region and / or an endogenous non-human 3' non-translated region are maintained are maintained in the modified non-human animal. Humanization of the non-human endogenous IL-15 locus may be with a coding region (or fragment thereof) which is a genomic fragment of a human IL-15 locus comprising at least one exon coding for human IL-15 protein. The humanization of the non-human endogenous IL-15 locus may be with a coding region which is a genomic fragment of a human IL-15 locus comprising each exon encoding human IL-15 protein, but which does not comprise an exon coding for non-human IL-15 protein. The IL-15 locus comprising the human genomic fragment may result in the expression of an IL-15 protein which, when mature, is fully human. Humanization of the non-human endogenous IL-15 locus may be with a cDNA encoding a human IL-15 protein such that, after processing in the non-human animal, the mature IL-15 protein produced by the humanized locus is fully human. A genetically modified non-human animal comprising an endogenous IL-15 locus is described which is humanized in whole or in part, wherein the humanized IL-15 locus comprises a gene encoding the humanized IL-15 that is under the control of endogenous non-human regulatory elements. Endogenous non-human regulatory elements may comprise all regulatory elements of the upstream endogenous IL-15 (with respect to the transcriptional sense of the IL-15 gene) of the first region or protein coding exon of the humanized IL-15 gene. Endogenous non-human regulatory elements may comprise all regulatory elements of the downstream endogenous IL-15 (with respect to the transcriptional sense of the IL-15 gene) of the last protein coding region or exon in the humanized IL-15 gene. The gene encoding humanized IL-15 may comprise a human 3'UTR. A genetically modified rodent comprising a substitution at an endogenous rodent IL-15 locus of an endogenous rodent IL-15 genomic sequence for a human IL-15 genomic sequence is described. The genetic modification may be in the germ line of the non-human animal. The genetically modified rodent may comprise a first upstream regulatory sequence (with respect to the sense of IL-15 gene transcription) of the human IL-15 genomic sequence and a second rodent regulatory sequence downstream of the IL-15 genomic sequence human. The first rodent regulatory sequence may comprise a rodent enhancer and / or enhancer, and the second rodent regulatory sequence comprises a 3'-UTR. A genetically modified nonhuman animal expressing a human or humanized IL-15 protein is described. A genetically modified mouse comprising a substitution at a mouse endogenous IL-15 locus of an endogenous mouse IL-15 genomic sequence (or fragment thereof) is described by a human IL-15 genomic sequence (or fragment thereof) to form a modified locus, wherein the genomic sequence of human IL-15 comprises at least one exon coding for human protein.

The substitution may comprise a human genomic fragment comprising at least two exons encoding human IL-15 protein. The substitution may comprise a human genomic fragment comprising at least three exons encoding human IL-15 protein. The substitution may comprise a human genomic fragment comprising at least four exons encoding human IL-15 protein. The substitution may comprise a human genomic fragment comprising exons 3, 4, 5 and 6 encoding human IL-15 protein. The substitution may comprise less than all exons of human IL-15, wherein the human exons of the substitution consist of the four exons coding for protein mars downstream (with respect to the sense of the transcription of the IL-15 gene) of the IL gene -15. The substitution may essentially consist of a human genomic fragment containing no more than four exons encoding human IL-15 protein. The substitution may further essentially consist of a human intron sequence upstream of the human exon closest to 5 'and in a human protein non-coding sequence downstream of the human stop codon and downstream of the 3' human UTR. A genetically modified mouse comprising a humanized IL-15 locus, wherein the humanized IL-15 locus comprises non-protein coding mouse exons, wherein each mouse exon coding for (mature) protein is replaced with exons protein (mature) coding proteins. The humanized IL-15 locus may comprise a replacement of a mouse genomic fragment encoding sequences of the mature (ie not pre-protein) mature IL-15 protein by a human genomic fragment encoding IL-15 protein sequences (ie, not pre-protein). A genetically modified mouse comprising a sequence that is at least 95%, 96%, 97%, 98%, or 99% identical or identical to SEQ ID NO: 5 is described. A genetically modified mouse comprising a sequence that is at least 95%, 96%, 97%, 98%, or 99% identical or identical to SEQ ID NO: 5 is described; wherein the mouse lacks an endogenous sequence encoding the 3 through 6 exons of a mouse IL-15 protein as depicted herein, and the mouse comprises a nucleic acid sequence at a locus of the endogenous mouse IL-15, wherein the nucleic acid sequence encodes exons 3, 4, 5 and 6 of human IL-15, as depicted herein. A genetically modified rodent expressing a human or humanized IL-15 protein from an endogenous mouse IL-15 locus is disclosed which is modified to comprise at least one exon of human IL-15 encoding amino acids in an IL- 15. In one embodiment, the locus of the endogenous rodent IL-15 comprises at least two exons of human IL-15 encoding amino acids in a mature human IL-15 protein. The locus of endogenous rodent IL-15 may comprise at least three exons of human IL-15 encoding amino acids in a mature human IL-15 protein. The locus of endogenous rodent IL-15 may comprise at least four exons of human IL-15 encoding amino acids in a mature human IL-15 protein. The endogenous rodent comprises exons 3, 4, 5 and 6 of human IL-15 encoding amino acids in a mature human IL-15 protein. The locus of endogenous rodent IL-15 may comprise the entire human nucleic acid sequence encoding amino acids in a mature human IL-15 protein. Humanization may comprise a 3'UTR of human IL-15. The rodent locus may comprise at least one non-amino acid coding exon of a mature IL-15 protein or at least one exon including a nucleotide sequence that does not encode amino acids of a mature IL-15 protein. The rodent locus may comprise at least two exons that do not encode amino acids from a mature IL-15 protein. The rodent locus may comprise at least three exons that do not encode amino acids of a mature IL-15 protein. The rodent locus may comprise four exons which do not encode amino acids from a mature IL-15 protein. A genetically modified rodent expressing a humanized IL-15 protein is described, wherein the rodent lymphocyte population is characterized by its T cell population having about the same number as a T cell population in a wild-type matched mouse age. The modified rodent may be characterized by a population of mature T cells having about the same number as a population of mature T cells in an age-matched wild type mouse. A genetically modified rodent expressing a humanized IL-15 protein, wherein the rodent lymphocyte population is characterized by a population of T cells having about the same number as a population of T cells in a wild-type mouse matched by age. The modified rodent may exhibit a mature T cell population having about the same number as a mature T cell population in an age-matched wild type mouse. The modified rodent can exhibit a population of peripheral T cells having about the same number as the population of peripheral T cells in an age-matched wild type mouse. The mature humanized IL-15 protein may be identical to a mature human IL-15 protein. A genetically modified rodent expressing a humanized IL-15 protein is described, wherein the rodent lymphocyte population is characterized by a T cell population exhibiting a CD4: CD8 ratio that is approximately equal to the CD4: CD8 ratio in T-cell population of an age-matched wild type mouse. The humanized IL-15 protein may be identical to a human IL-15 protein.

A genetically modified rodent expressing a humanized IL-15 protein is described, wherein the rodent lymphocyte population is characterized by a natural killer (NK) cell population that is about the same size as a NK cell population in a mouse of the wild type matched for age. The humanized IL-15 protein may be identical to a human IL-15 protein. A genetically modified rodent expressing a human or humanized IL-15 protein, wherein the rodent lymphocyte population is characterized by a population of T cells and a population of NK cells each having about the same size as a population of T cells and a population of NK cells in an age-matched wild type mouse. A genetically modified rodent expressing a humanized IL-15 protein is described, wherein the rodent does not develop spontaneous intestinal inflammation. The rodent may not have a propensity to develop intestinal inflammation more than an age-matched wild type rodent. A genetically modified rodent expressing a humanized IL-15 protein is described, wherein the rodent does not develop spontaneous inflammation in the duodeno-jejunal area. The rodent may not have a propensity to develop inflammation in the duodenum-jejunal area more than an age-matched wild type rodent. A genetically modified rodent expressing a humanized IL-15 protein, wherein the rodent does not exhibit destruction of the intestinal epithelium is described more than an age-matched wild type rodent. A genetically modified rodent expressing a humanized IL-15 protein is described, wherein the rodent does not exhibit celiac disease at a rate or frequency higher than an age-matched wild type rodent. A genetically modified rodent expressing a humanized IL-15 protein, wherein the rodent does not exhibit higher resistance to diet-induced adiposity, and does not exhibit a higher insulin sensitivity, is described than a wild-type rodent paired by age. A genetically modified rodent expressing a humanized IL-15 protein is described, wherein the rodent, upon infection with a pathogen, is no longer susceptible to lipopolysaccharide-induced lethal liver injury than an age-matched wild type rodent. A genetically modified rodent expressing a humanized IL-15 protein, wherein the rodent does not develop psoriatic lesions at a rate or frequency higher than an age-matched wild type rodent, is described. A genetically modified rodent expressing a humanized IL-15 protein, wherein the rodent does not develop arthritis at a rate or frequency higher than an age-matched wild type rodent, is described. A genetically modified rodent expressing a humanized IL-15 protein is described, wherein the rodent does not develop lymphocytic infiltration of the joints at a rate or frequency higher than an age-matched wild type rodent. A genetically modified rodent expressing a humanized IL-15 protein is described, wherein the rodent does not develop inflammatory synovitis at a higher rate than an age-matched wild type control rodent. A genetically modified rodent expressing a human or humanized IL-15 protein, wherein the rodent does not develop one or more conditions at a rate or frequency higher than that of an age-matched wild type rodent, is described. or more pathologies are selected from the group consisting of arthritis, lymphocytic infiltration of the joints, inflammatory synovitis, psoriatic lesions, lethal hepatic injury induced by pathogen-related lipopolysaccharide, insulin resistance, resistance to dietary adiposity, spontaneous intestinal inflammation, inflammation spontaneous in the duodenum-jejunal area, destruction of the intestinal epithelium, celiac disease, and a combination thereof. A genetically modified rodent expressing a human or humanized IL-15 protein is described, wherein the rodent does not develop two or more conditions at a rate or frequency higher than an age-matched wild type rodent, wherein the two or more pathologies are selected from the group consisting of arthritis, lymphocytic infiltration of the joints, inflammatory synovitis, psoriatic lesions, lethal hepatic injury induced by pathogen-related lipopolysaccharide, insulin resistance, dietary resistance to adiposity, spontaneous inflammation of the intestine, spontaneous inflammation in the duodeno-jejunal area, destruction of the intestinal epithelium, and celiac disease. A genetically modified rodent expressing a human or humanized IL-15 protein is described, wherein the rodent does not develop three or more pathologies at a rate or frequency higher than that of an age-matched wild type rodent, wherein the three or more pathologies are selected from the group consisting of arthritis, lymphocytic infiltration of the joints, inflammatory synovitis, psoriatic lesions, lethal hepatic injury induced by pathogen-related lipopolysaccharide, insulin resistance, resistance to dietary adiposity, spontaneous intestinal inflammation, inflammation spontaneous in the duodenum-jejunal area, destruction of the intestinal epithelium, and celiac disease. A genetically modified rodent expressing a human or humanized IL-15 protein is described, wherein the rodent does not develop four or more pathologies at a rate or frequency higher than an age-matched wild type rodent, wherein the four or more pathologies are selected from the group consisting of arthritis, lymphocytic infiltration of the joints, inflammatory synovitis, psoriatic lesions, lethal hepatic injury induced by pathogen-related lipopolysaccharide, insulin resistance, dietary resistance to adiposity, spontaneous inflammation of the intestine, spontaneous inflammation in the duodeno-jejunal area, destruction of the intestinal epithelium, and celiac disease. A genetically modified rodent expressing a human or humanized IL-15 protein is described, wherein the rodent does not develop five or more pathologies at a rate or frequency higher than an age-matched wild type rodent, wherein the five or more pathologies are selected from the group consisting of arthritis, lymphocytic infiltration of the joints, inflammatory synovitis, psoriatic lesions, lethal hepatic injury induced by pathogen-related lipopolysaccharide, insulin resistance, dietary resistance to adiposity, spontaneous inflammation of the intestine, spontaneous inflammation in the duodeno-jejunal area, destruction of the intestinal epithelium, and celiac disease. A genetically modified rodent expressing a human or humanized IL-15 protein is described, wherein the rodent does not develop six or more pathologies at a rate or frequency higher than an age-matched wild type rodent, wherein the six or more pathologies are selected from the group consisting of arthritis, lymphocytic infiltration of the joints, inflammatory synovitis, psoriatic lesions, lethal hepatic injury induced by pathogen-related lipopolysaccharide, insulin resistance, dietary resistance to adiposity, spontaneous inflammation of the intestine, spontaneous inflammation in the duodenum-jejunal area, destruction of the intestinal epithelium, and celiac disease. A genetically modified animal comprising a humanization of an endogenous IL-15 locus, wherein the animal expresses a partially or fully human mature IL-15 protein, and wherein the partially or fully human mature IL-15 protein is expressed at comparable levels and in the same tissues as an endogenous IL-15 protein in an age-matched wild-type animal. A large targeting vector (LTVEC) comprising homology arms at a non-human endogenous IL-15 locus is described, wherein the LTVEC comprises, between said homology arms, a contiguous human genomic fragment comprising codons a human IL-15 gene. The contiguous human genomic fragment may not comprise non-coding exons of a human IL-15 locus protein. The contiguous human genomic fragment may further comprise a human 3'UTR of an IL-15 gene. A nucleic acid construct (for example, DNA) is described which comprises, from 5 'to 3', with respect to the direction of transcription, a nucleic acid sequence homologous to a 5 'non-coding sequence of mouse IL-15 , a human genomic fragment comprising exons encoding human IL-15 protein, but which do not comprise a human regulatory sequence upstream of the human IL-15 protein coding sequence, and a nucleic acid sequence homologous to a non-coding sequence in 3 'cells of mouse IL-15. The human genomic fragment may further comprise a 3'UTR of human IL-15. A nucleic acid construct (e.g., DNA) is described which comprises, from 5 'to 3', with respect to the direction of transcription, a nucleic acid sequence comprising a region of homology in the IL-15 gene sequences of mouse upstream of the first codon exon of the mouse IL-15 protein, a human genomic fragment encoding a human IL-15 protein, but which does not comprise a human regulatory sequence upstream of the sequence encoding the human IL-15 protein, and a nucleic acid sequence homologous to a 3 'non-coding sequence of mouse IL-15. The human genomic fragment may further comprise a 3'UTR of human IL-15. A non-human genetically modified cell, wherein the non-human cell comprises a substitution at a non-human endogenous IL-15 locus of a gene sequence encoding a non-human IL-15 by a human genomic sequence encoding a Human IL-15. The human genomic sequence may comprise a contiguous human nucleic acid sequence encompassing all the exons encoding human IL-15 gene protein. The genetically modified rodent may comprise a non-human IL-15 promoter at the non-human endogenous IL-15 locus. The genetically modified nonhuman animal may comprise all non-coding rodent exons and regulatory regions upstream of the first codon exon protein of the rodent IL-15 gene. The cell may be selected from a pluripotent cell, an induced pluripotent cell, a totipotent cell, an ES cell, a somatic cell, and an ovule. The cell may express the human IL-15 protein.

Non-human animals may be a mammal. The mammal may be a rodent. The rodent can be selected from a mouse and a mouse. A non-human embryo is described, wherein the embryo comprises at least one non-human donor cell (e.g., an ES cell, a pluripotent cell, a totipotent cell, etc.) comprising a substitution of a nucleic acid sequence encoding IL -15 by a nucleic acid sequence encoding human IL-15 at a non-human endogenous IL-15 locus. The donor cell may be a non-human ES cell and the embryo is a host non-human animal embryo that is a pre-morula, a morula, or a blastocyst. The non-human embryo may be a mouse embryo, and the at least one non-human donor cell is a mouse cell. The non-human ES cell may be a rat ES cell and the host embryo is a mouse embryo. The non-human embryo may be a mouse embryo, and the at least one non-human donor cell is a mouse cell. The non-human ES cell may be a mouse ES cell and the host embryo is a mouse embryo. A rodent tissue comprising a humanized IL-15 gene is described in an endogenous rodent IL-15 locus. The tissue may be selected from epithelial tissue, skin tissue, and muscle tissue. A genetically modified rodent is described which comprises a nucleic acid sequence (e.g., DNA) encoding a human IL-15 protein, wherein the rodent does not express a rodent IL-15, and wherein the rodent exhibits a population of NK cells that are about the same size as a NK cell population of a wild type rodent. The rodent can be a mouse. The rodent can be a mouse. The rodent can exhibit a population of peripheral T cells that is approximately the same size as a population of peripheral T cells from an age-matched wild type rodent. A method of producing a non-human animal expressing a human or humanized IL-15 protein, comprising the genetic modification of a non-human animal as described herein, is described to form a nucleic acid sequence in the non-human animal comprising a nucleic acid sequence (e.g., DNA) encoding a human or humanized IL-15 protein, wherein the nucleic acid sequence is under the control of non-human endogenous upstream and downstream regulatory elements.

The non-human animal can be genetically modified by replacing an endogenous IL-15 genomic sequence (or fragment thereof) at an endogenous IL-15 locus by a human IL-15 genomic sequence (or fragment thereof) to form a modified locus, wherein the genomic sequence of human IL-15 comprises at least one exon encoding human protein. The substitution may comprise a human genomic fragment comprising at least two exons encoding human IL-15 protein. The substitution may comprise a human genomic fragment which comprises at least three exons encoding human IL-15 protein. The substitution may comprise a human genomic fragment comprising at least four exons encoding human IL-15 protein. In a specific embodiment, the substitution comprises a human genomic fragment comprising exons 3, 4, 5 and 6 encoding human IL-15 protein. The substitution may further comprise a non-coding human protein sequence downstream of the human stop codon (e.g., human 3'UTR). The non-human animal may be produced from a pluripotent or totipotent cell (e.g., an ES cell). The non-human animal can be produced by the use of a nuclear injection step, wherein a nucleic acid construct comprising the humanized IL-15 gene (optionally with upstream and / or downstream endogenous non-human regulatory sequences) is introduced by pro-nuclear injection. The nucleic acid construct may comprise a human genomic fragment comprising exons 3, 4, 5 and 6 encoding human IL-15 protein. The nucleic acid construct may further comprise a human protein non-coding sequence downstream of the human stop codon (e.g., human 3'UTR). The non-human animal can be produced by employing a non-human fibroblast that is genetically modified with a human or humanized IL-15 gene and (optionally) upstream and / or downstream non-human IL-15 regulatory elements. A method of identifying an agent that is an antagonist of human IL-15, comprising a step of administering an agent to a genetically modified rodent, as described herein, determining an effect of the agent on an IL-mediated function -15 in the rodent, and the identification of the agent as an antagonist of IL-15 if it antagonizes the function of human IL-15 in the genetically modified rodent. The agent may comprise an immunoglobulin variable domain which binds to IL-15. The agent may specifically bind to human IL-15, but not to rodent IL-15. The agent may be an antibody. A method is described for determining whether an agent reduces IL-15 mediated lymphocyte development, comprising a step of administering to a genetically modified rodent, as described herein, an IL-15 antagonist for a period of time, measuring a number of rodent lymphocytes at one or more times post-administration, and determining whether the IL-15 antagonist reduces the lymphocyte population. A method is described for determining whether an agent reduces IL-15 mediated lymphocytic infiltration of a tissue or a joint, comprising a step of administering to a genetically modified rodent, as described herein, an IL-15 antagonist by a time period, measuring lymphocytic infiltration of the tissue or joint at one or more times after administration, and determining whether the IL-15 antagonist reduces lymphocytic infiltration of the tissue or the joint. A method is described for determining whether an agent reduces IL-15 mediated arthritic progression, comprising a step of administering to a genetically modified rodent, as described herein, and further comprising an induced arthritis, an IL-15 antagonist for a time period, measurement of arthritic progression, and determination of whether the IL-15 antagonist affects arthritic progression in the rodent.

Unless otherwise noted, or evident from the context, two or more aspects and / or modalities may be combined.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 depicts (non-scaled) a scheme of a (superior) locus of the wild-type mouse IL-15 gene and a (lower) locus of the humanized endogenous mouse IL-15. Open symbols indicate a human sequence; closed symbols indicate a mouse sequence; dotted items indicate untranslated regions. The non-coding exons (left to right in the figure) of the mouse IL-15 gene are not shown (and not humanized). The lower construct represents a modality of a humanized IL-15 gene comprising a humanized (dotted) 3'UTR and a removable drug screening cassette, which is optionally removable in the humanized animal. FIG. 2 is a nucleic acid sequence (SEQ ID NO: 1) representing the upstream junction (in respect of the sense of transcription of the IL-15 gene) between the mouse sequence and the human sequence; the sequence shown begins with the mouse sequence in block letters, followed by an Asisl restriction site in lower case, followed by the nucleic acid sequence of human IL-15 in block letters. Ellipses indicate that the sequence continues upstream and downstream of the sequence shown. FIG. 3 is an embodiment of a nucleic acid sequence (SEQ ID NO: 2) which indicates a coding and non-coding sequence of human IL-15 downstream in human capital letters 3'UTR underlined), followed by a low-order Xhol site , followed by a lox site (in capital letters, underlining), followed by the sequence of the neo downstream selection cassette (in block letters), which extends 2.6 kb downstream. FIG. 4 is a nucleic acid sequence (SEQ ID NO: 3) which represents the junction between the downstream portion of the neo selection cassette (in block letters), with lox site (in capital letters and underlining), followed by a Nhel site (in lower case), which is followed by the mouse sequence downstream of humanization (in block letters); the 2.6 kb indicates that the selection cassette extends further upstream; the ellipses indicate that the sequence continues. FIG. 5 depicts an alignment of the protein sequences for the mouse precursor protein IL-15 (superior, "precursor mIL15," SEQ ID NO: 4); the hybrid mouse / human IL-15 precursor protein (medium, "Regn_Hybrid_m / h," SEQ ID NO: 5) produced by a humanisation of the locus depicted in FIG. 1; and the known human IL-15 isoform 1 (lower, "hIL15 isoform 1," SEQ ID NO: 6) pre-protein; the resulting junction of the humanization, as shown in FIG. 1 is indicated by a vertical arrow indicating C as the first amino acid of the substitution; the onset of the mature hIL-15 protein is indicated by the curved arrow on the first N-amino acid. FIG. 6 depicts the IL-15 detected in the serum of heterozygote hIL-15 mice injected with poly I: C. Control indicates that no h-L-15 was detected in splenocytes in culture of wild type mice injected with poly I: C. FIG. 7 depicts that human IL-15 does not react with either mouse IL-15 or poly I: C in an ELISA assay. The mouse IL-15 was at 1000 pg / ml. FIG. 8 depicts BM-DCs from heterozygous mice to human IL-15, using 7-fold concentrated BM-DC supernatants to untreated mice, poly I: C treated mice, and LPS treated mice. FIG. 9 shows that BM-DCs from heterozygous mice to human IL-15 produce the transcript of human IL-15 (RT-PCR data). Lane 1: untreated mice; Lane 2: mice treated with poly I: C; Track 3: only with LPS; Lane 4: untreated wild-type mice; Lane 5: wild-type mice treated with poly I: C; Lane 6: LPS-treated wild type mice; Track 7: no cDNA control (water only). FIG.10 shows that het MAID 5218 mice produced human IL-15 after injection of poly I: C at a level comparable to het MAID 5217 mice.

DETAILED DESCRIPTION Non-human genetically modified organisms comprising a modified endogenous IL-15 locus comprising a human sequence, for example, a substitution of one or more non-human sequences for one or more human sequences are described. The organisms are generally capable of passing the modification to the progeny, that is, through transmission through the germ line. In particular, non-human genetically modified animals are described.

Genetically modified non-human animals may be selected from a group consisting of a mouse, rabbit, pig, bovine (e.g., cow, bull, buffalo), deer, sheep, goat, chicken, cat, ferret, primates ( for example, sago, rhesus monkey). For non-human animals in which suitable genetically modifiable ES cells are not readily available, other methods are employed to produce a non-human animal comprising the genetic modification. Such methods include, for example, modification of a non-ES cell genome (e.g., a fibroblast or an induced pluripotent cell) and use of nuclear transfer to transfer the modified genome to a suitable cell, e.g., an oocyte, and gestation of the modified cell (e.g., modified oocyte) in a non-human animal under conditions suitable to form an embryo.

Non-human animals may be a mammal. The non-human animal may be a small mammal, for example, of the superfamily Dipodoidea or Muroidea. In one embodiment, the genetically modified animal is a rodent. The rodent can be selected from a mouse, a mouse and a hamster. The rodent can be selected from the superfamily Muroidea. The genetically modified animal may be from a family selected from Calomyscidae (e.g., mouse-like hamsters), Cricetidae (e.g., hamsters, New World mice and rats, wild mice), Muridae (true mice and rats, gerbils, spiny rats), Nesomyidae (climbing mice, rock mice, rats with cause, mice and Malagasy mice), Platacanthomyidae (e.g., spiny argan), and Spalacidae (e.g., mole rats, rats bamboo and zokors). The genetically modified rodent may be selected from a true mouse or mouse (Muridae family), a gerbil, a spiny mouse, and a crested mouse. The genetically modified mouse may be one of a member of the Muridae family. The animal may be a rodent. The rodent can be selected from a mouse and a mouse. The non-human animal may be a mouse.

In a specific embodiment, the mouse is from a C57BL / A, C57BL / An, C57BL / GrFa, C57BL / KaLwN, C57BL / 6, C57BL / 6J, C57BL / 6ByJ, C57BL / 6NJ, C57BL / C57BL / 1OScSn, C57BL / 10Cr, and C57BL / 01a cells. In another embodiment, the mouse is a race selected from the group consisting of a race which is 129P1, 129P2, 129P3, 129X1, 129S1 (e.g., 129S1 / SV, 129S1 / SvIm), 129S2, 129S4, 129S5, 129S9 / SvEvH, (See, for example, Festing et al. (1999) Revised nomenclature for strain 129 mice, Mammalian Genome 10: 836, see also, Auerbach et al. (2000) Establishment and Chimera Analysis of 129 / SvEv- and C57BL / 6-Derived Mouse Embryonic Stem Cell Lines). In a specific embodiment, the genetically modified mouse is a mixture of a previously mentioned race and a C57BL / 6 race mentioned above. In another specific embodiment, the mouse is a mixture of a race mentioned above, or a mixture of a BL / 6 race mentioned above. In a specific embodiment, the 129 race of the mixture is 12 9S6 race (129 / SvEvTac). In another embodiment, the mouse is a BALB race, e.g., BALB / c race. In yet another embodiment, the mouse is a mixture of a BALB and other race mentioned above. In yet another embodiment, the mouse is from a hybrid strain (for example, 50% BALB / c-50% 129S4 / Sv; or 50% C57BL / 6-50% 129; for example, F1H4 cells, see, for example, Auerbach et al (2000)). The nonhuman animal can be a mouse. The mouse can be selected from among a Wistar rat, a LEA breed, a Sprague Dawley breed, a Fischer breed, F344, F6, and Dark Agouti. The rat race may be a mixture of two or more races selected from the group consisting of Wistar, LEA, Sprague Dawley, Fischer, F344, F6, and Dark Agouti. The non-human animal may have one or more other genetic modifications, and / or other modifications, that are suitable for the specific purpose for which the humanized IL-15 mouse is made. For example, mice suitable for maintaining a xenograft (e.g., a cancer or human tumor) may comprise one or more modifications that compromise, inactivate or destroy the immune system of the non-human animal in whole or in part. The impairment, inactivation or destruction of the immune system of the non-human animal may include, for example, the destruction of hematopoietic cells and / or immune cells by chemical means (e.g., administration of a toxin), physical means (e.g. animal), and / or genetic modification (eg, knockout for one or more genes). Non-limiting examples of such mice may include, for example, NOD mice, SCID mice, NON / SCID mice, knockout mice for IL2Ry, NOD / SCID / Ycnu11 mice (see, e.g., Ito, M. et al. (2002) NOD / SCID / Ycnu11 mouse: an excellent recipient mouse model for engraftment of human cells, Blood 100 (9): 3175-3182), nude mice, and knockout mice for Ragl and / or Rag2. Such mice may optionally undergo radiation, or otherwise be treated to destroy one or more types of immune cells. Thus, a genetically modified mouse is described which comprises a humanization of at least a portion of a non-human endogenous IL-15 locus, and further comprises a modification that compromises, inactivates or destroys the immune system (or one or more of the cell types of the immune system) of the non-human animal in whole or in part. The modification is, for example, selected from the group consisting of a modification that results in a NOD mouse, a SCID mouse, a NOD / SCID mouse, a knockout mouse for IL-2Ry, a NOD / SCID / yc1 ™ 11 mouse, a mouse mouse nude, a mouse knockout for Ragl and / or Rag2, and a combination thereof. The mouse may comprise a replacement of the entire mature IL-15 coding sequence by a coding sequence of mature human IL-15. The mouse may comprise a replacement of the entire mature IL-15 coding sequence by a coding sequence of mature human IL-15, and the mouse is a NOD mouse. The mouse may comprise a replacement of the entire mature IL-15 coding sequence by a coding sequence of mature human IL-15, and the mouse is a SCID mouse. The mouse may comprise a replacement of the entire mature IL-15 coding sequence by a coding sequence of mature human IL-15, and the mouse is a NOD / SCID mouse. The mouse may comprise a replacement of the entire mature IL-15 coding sequence by a coding sequence of mature human IL-15, and the mouse comprises a knockout for IL-2Ry. The mouse may comprise a replacement of the entire mature IL-15 coding sequence by a coding sequence of mature human IL-15, and the mouse is a NOD / SCID / Ycnu11 mouse. The mouse may comprise a replacement of the entire mature IL-15 coding sequence by a coding sequence of mature human IL-15, and the mouse is a nude mouse. The mouse may comprise a replacement of the entire mature IL-15 coding sequence by a coding sequence of mature human IL-15, and the mouse comprises a knockout for Ragl and / or Rag2. Genetically modified non-human animals comprising a modification of a non-human endogenous IL-15 locus are described, wherein the modification comprises a human nucleic acid sequence encoding at least a portion of a mature IL-15 protein. Although genetically modified cells comprising the modifications described herein (e.g., ES cells, somatic cells) are also described, non-human genetically modified animals may comprise modification of the locus of endogenous IL-15 in the germ line of the animal. Non-human genetically modified animals comprising a replacement of a non-human IL-15 gene sequence by a human IL-15 gene sequence are described. A non-human endogenous IL-15 locus may be modified in whole or in part to comprise a human nucleic acid sequence encoding at least one protein coding exon of a mature IL-15 protein. The human sequence may be a human genomic sequence, for example, a contiguous human genomic sequence comprising one or more exons encoding a portion of a mature IL-15 protein, or, for example, a cDNA encoding at least one or more exons which encode a portion of a mature IL-15 protein. All exons encoding the IL-15 protein encoding protein sequences appearing in a mature human IL-15 protein can be humanized. The locus of the humanized IL-15 may be under the control of upstream regulatory sequences (for example, all endogenous sequences upstream of the humanization). Humanization may comprise a human 3'UTR.

Nonhuman animals may be mammals. Mammals can be rodents. Rodents are described which comprise a humanization of an IL-15 gene at an endogenous rodent IL-15 locus. Methods for producing rodents are described, for example, mice comprising a replacement of an endogenous IL-15 gene or a fragment thereof (e.g., a fragment comprising one or more exons) by a humanized IL-15 gene, or a fragment thereof (e.g., a fragment comprising one or more exons) at the endogenous IL-15 locus. Cells, tissues and mice comprising the humanized gene, as well as cells, tissues and mice expressing the humanized IL-15 of a non-human endogenous IL-15 locus are described. Rodents expressing a human IL-15 protein under the control of an endogenous rodent promoter are also described. IL-15 has been discovered as an IL-2-independent T cell growth factor that stimulates T cell proliferation and supports thymic development and the development of natural killer (NK) cells (Burton, JD et al., 1994) A lymphokine, provisionally designated interleukin T and produced by a human adult T-cell leukemia line, stimulates T-cell proliferation and the induction of lymphokine-activated killer cells, Proc. Natl. Acad Sci USA 91: 4935-4939; Grabstein , KH et al. (1994) Cloning of a T Cell Growth Factor That Interacts with the β Chain of the Interleukin-2 Receptor, Science 264: 965-968). IL-2 and IL-15 share receptor subunits. However, the independent importance of IL-15 in the maintenance of immune cell populations is not disputed; knockout mice for IL-15 / IL-15R exhibit few CD8 + T cells, few memory CD8 + T cells, and few NK, as well as other cell types (reviewed in Steel, JC et al. (2012) Interleukin-15 biology and its therapeutic implications in cancer, Trends in Pharmacological Sciences, 33 (1): 35-41). IL-15 is known to be expressed in endothelial cells; IL-15 derived from endothelial cells stimulates transendothelial migration of T cells (see, Oppenheimer-Marks, N. (1998) Interleukin 15 is Produced by Endothelial Cells and Increases the Transendothelial Migration of T Cells in Vitro and in the SCID Mouse- Human Rheumatoid Arthritis Model In Vivo, J. Clin. Invest 101 (6): 1261-1272). Thus, an initial study established a probability that recruitment of T cells to inflammatory sites be mediated by IL-15 (Id.). This is significant because overexpression or improper expression of IL-15 can readily lead to a pathological phenotype - a situation faced with non-human transgenic animals expressing deregulated IL-15. Proper regulation of IL-15 is important because IL-15 is believed to be a proinflammatory cytokine that is at the apex of a cascade of proinflammatory cytokines, preceding the expression of many inflammatory mediators (Mclnnes, IB et al. (1997) Interieukin-15 mediates T cell-dependent regulation of tumor necrosis factor-α production in rheumatoid arthritis, Nature Med. 3: 189-195, quoted in Waldmann, TA (2006) The biology of interleukin-2 and interleukin- 15: implications for cancer therapy and vaccine design, Nature Rev. Immun. 6: 595-601).

Transgenic mice expressing human IL-15 under the control of an enterocyte-specific promoter (T3b promoter) to express human IL-15 in intestinal epithelial cells develop spontaneous inflammation in the duodeno-jejunal area (Yokoyama, S. et al. 2008) Antibody-mediated blockade of IL-15 reverses the autoimmune intestinal damage in transgenic mice that overexpress IL-15 in enterocytes, Proc Natl Acad Sci USA 106 (37) 15849-15854, Ohta, N. et al. (2002) IL-15-dependent activation-induced cell death-resistant Thl type CD8 alpha beta + NK1.1 + T cells for the development of small intestinal inflammation, J. Immunol 169: 460-468). See, also, Nishimura, H. et al. (2005) A novel autoregulatory mechanism for transcriptional activation of IL-15 gene by nonsecretable isoform of IL-15 generated by alternative splicing, FASEB J. 19: 19-28 (transgenic mice with a variant of the randomly inserted mIL-15 gene ).

Transgenic mice for a secretable isoform of IL-15 under the control of a class 1 MHC promoter were prepared, but they overexpressed IL-15 (Yajima, T. et al. (2001) Memory phenotype CD8 (+) T cells in IL-15 transgenic mice are involved in early protection against primary infection with Listeria monocytogenes, Eur.J. Immunol. 31 (3): 757-766). The overexpression of IL-15 is correlated with the destruction of the intestinal epithelium by IL-15-activated cytotoxic T lymphocytes in celiac disease (Yokoyama, S. et al. (2011) Transgenic Mice that Overexpress Human IL-15 in Enterocytes Recapitulate Both B and Cell-Mediated Pathologic Manifestations of Celiac Disease, J. Clin Immunol 31: 1038-1044), presumably due to the promotion of the proliferation of enterocyte-targeting CD8 + T cells through the NKG2D-mediated process (natural killer group 2, member D) comprising cognate receptors, such as MICA / B (Id., 1039). It now appears clear that locally expressed IL-15 causes T-cell mediated tissue damage in the gut in celiac disease (Id.).

At least one study of transgenic mice that are designed to overexpress IL-15 in muscle tissue and circulation (employing a skeletal muscle promoter) states that IL-15 overexpression affects metabolism; these mice appear to employ IL-15 as a myocin that reduces body fat and provides resistance to diet-induced adiposity (Quinn, LS et al. (2009) Oversecretion of interleukin-15 from skeletal muscle reduces adiposity, Am. J. Physiol Endocrinol Metab 296: E191-E202).

IL-15 is also thought to be involved in rheumatoid arthritis, perhaps through abnormal infiltration of T cells into the joints (reviewed, for example, in Fehninger TA and Caligiuri, MA (2001) Interleukin 15: biology and relevance to human disease , Blood 97 (1): 14-32). Patients with sarcoidosis also produce alveolar macrophages expressing IL-15, which can mediate T-cell proliferation in the lung (Id., At 23). IL-15 can also mediate organ rejection in allografts through T-cell proliferation (Id., At 24). IL-15 may also be implicated in adult (e.g., HTLV-1 mediated) T cell leukemia based, at least in part, on the activation of IL-15 mediated pathways in patients with T-cell leukemia of adult (Id.). In vitro work suggests that IL-15 activates HIV replication, which may also be the case in humans (Id., At 25).

In transgenic mice expressing IL-15 driven by a class I MHC promoter infected with Mycobacterium bovis Calmette-Guérin, overproduction of IL-15 rendered mice susceptible to LPS-induced lethal liver injury, an effect that was not observed when CD8 + T cells were destroyed from the mice (Yajima, T. (2004) Overexpression of Interleukin-15 Increases Susceptibility to Lipopolysaccharide-Induced Liver Injury in Mice Primed with Mycobacterium bovis Bacillus Calmette-Guérin, Infection and Immunity 72 (7): 3855 -3862), suggesting an effect mediated by IL-15 overproduction.

Transgenic mice expressing IL-15 driven by a skeletal muscle promoter exhibited increased insulin sensitivity and diet-induced obesity resistance, and appeared to promote fatty acid metabolism (Quinn, LS et al. (2011) Overexpression of interleukin-15 in mice promotes resistance to diet-induced obesity, increased insulin sensitivity, and markers of oxidative skeletal muscle metabolism, International Journal of Interferon, Cytokine and Mediator Research, 3: 29-42). Selective blocking of murine IL-15 has been studied, including a soluble IL-15Ra, which may have clinical benefit in the control of rheumatoid arthritis (Id., At 27). Thus, non-human animals expressing human or humanized IL-15, including in a physiologically relevant manner, are useful for evaluating or identifying selective human IL-15 blockers. According to one reviewer, "the development of effective human IL-15 blocking agents ... with in vivo blocking activity could facilitate the rapid translation of these approaches into the clinic" (Id., At 27). Thus, genetically modified non-human animals, for example, non-human animals comprising a human IL-15 gene in their germ line, where non-human animals express the human IL-15 in a physiologically suitable manner, would be very useful. IL-15 is a pleiotropic cytokine that is required for the development and function of NK cells and T-cell homeostasis. It is particularly important for the memory CD8 + T cell compartment. IL-15 is produced primarily by dendritic cells and macrophages, and is transpresented through the IL-15 / 1L-15R complex to NK cells and T cells. IL-15 is also known to be a proinflammatory cytokine that induces the production of other cytokines, recruits and activates T cells and other inflammatory cells, promotes the development and survival of NK cells and promotes angiogenesis; and many of these characteristics are presented in psoriatic lesions (reviewed and reported in Villadsen, LS (2003) Resolution of psoriasis upon blockade of IL-15 biological activityin a xenograft mouse model, J. Clin. Invest. 112 (10): 1571-1580 ). It has been proposed that IL-15 is at the apex of the cascade of proinflammatory cytokines, with various strategies to modulate IL-15 signaling for the treatment of disease (reviewed in Waldman (2006) The biology of interleukin-2 and interleukin- 15: implications for cancer therapy and vaccine design, Nature Reviews Immunology, 6: 595-601). Thus, in a mouse xenograft model of SCID mice, blocking IL-15 using an antibody to IL-15R (or IL-15) resulted in a reduction in the severity of psoriasis (Id.). Thus, non-human animals expressing physiologically relevant IL-15 are useful (for example, have a well-established utility) in models for human diseases, including, but not limited to, models in immunocompromised mice such as SCID mice and other immunocompromised mice. Thus, a rodent (e.g., a mouse) is described which comprises a human or humanized IL-15 gene under the control of non-human endogenous regulatory elements (e.g., a humanization of the gene coding for IL-15 in a rodent , for example, a mouse). The IL-15 gene is found on human chromosome 4q31 and mouse chromosome 8. The human gene contains 8 exons (7 coding) and appears to exist in two isoforms in humans and mice (see, for example, Fehninger TA and Caligiuri, MA (2001) Interleukin 15: biology and relevance to human disease, Blood 97 ): 14-32). IL-15 mRNA is produced in a wide variety of tissues and cell types and regulation of the IL-15 gene in humans appears to be upregulated by an upstream region whose deletion results in a dramatic increase in promoter activity of IL-15 (Id., at 17). Transgenic mice lacking post-transcriptional control of IL-15 present fatal lymphocytic leukemia (Id.). The regulation of IL-15 expression appears to be very fair, mediated at least triuns of the 5 'untranslated region, 3' regulatory elements and a putative C-terminal region regulatory site (reviewed in McInnes, IB and Gracie, JA ( 2004) Interleukin-15: a new cytokine target for the treatment of inflammatory diseases, Current Opinion in Pharmacology 4: 392-397). The human IL-15 gene has nine exons and eight introns, which includes an exon 4a that is present in humans but not in mice, although the mature IL-15 protein is encoded only by 5 to 8 exons (reviewed in Budagian, V. et al. (2006) IL-15 / IL-15 receptor biology: A guided tour through an expanding universe, Cytokine & Growth Factor Reviews 17: 259-280). There are two alternatively splice mRNA products that produce two IL-15 isoforms, which differ only in signal peptide length, which is true for mouse and human proteins (see, e.g., Id.). FIG. 1, which represents a humanization strategy for a mouse IL-15 locus, omits upstream mouse sequences that have not been humanized (including exons that do not appear in the mature protein) to maintain simplicity and present a renumbering of the relevant exons for the humanization shown. IL-15 is expressed in many cell types and tissues, including monocytes, macrophages, dendritic cells, keratinocytes, epidermal cells, fibroblasts and epithelial cells of the nerve, kidney, placenta, lung, heart and muscle (Grabstein, KH et al. (1994) Cloning of a T Cell Growth Factor That Interacts with the β Chain of the Interleukin-2 Receptor, Science 264: 965-968).

The mouse IL-15 coding sequences were humanized, as depicted in FIG. 1, which omits the representation of two non-coding (non-humanized) exons well upstream of the coding exons. 12299 nucleotides of the mouse sequence were replaced with 12896 nucleotides of human sequence to humanize the qene of IL-15. The humanized IL-15 locus does not have 5'UTR of human IL-15. The locus of the humanized IL-15 may comprise 5 'rodent UTR. The rodent can be a mouse and the humanized IL-15 locus comprises the 5'UTR of mouse IL-15.

Rodents expressing human or humanized IL-15 protein, for example, in a physiologically appropriate manner, provide a variety of uses including, but not limited to, the development of therapeutic agents for human diseases and disorders. IL-15 antagonists such as, for example, soluble or IL-15 receptor forms may prevent the development of collagen-mediated arthritis in an animal model (see, Ruchatz, H. et al., (1998) Soluble IL- 15 receptor α-chain administration prevents murine collagen-induced arthritis: a role for IL-15 in the development of antigen-induced immunopathology, J. Immunol 160: 5654-5660; the anti-IL-15 antibodies have shown efficacy against a variety of diseases, including psoriasis and rheumatoid arthritis; in an animal model of arthritis, an IL-15 receptor antagonist prevents the development and progression of arthritis, as well as reduces lymphocyte infiltration at the joints (Ferrari-Lacraz, S. et al. (2004) argeting IL-15 Receptor-Bearing Cells with an Antagonist Mutant IL-15 / Fc Protein Prevents Disease Development and Progression in Murine Collagen-induced Arthritis, J. Immunol 173: 5815-5826); IL-15-mediated signaling also implicated in IBD, SLE, inflammatory synovitis, diabetes mellitus and asthma (reviewed in Budagian, V. et al. (2006) IL-15 / IL-15 receptor biology: A guided tour through an expanding universe, Cytokine &

Growth Factor Reviews 17: 259-280).

Studies with knockout mice for IL-15 establish that IL-15 is required for the development of certain immune cells, in particular NK cells (reviewed in Lodoice, J.P. (2002)

Regulationof lymphoid homeostasis by interieukin-15, Cytokine & Growth Factor Reviews, 13: 429-439). In fact, knockout mice for IL-15 do not survive very long to certain pathogens (e.g., Vaccinia virus), presumably due to lack of NK cells and CD8 + T (Id.). Thus, the effects of hIL-15 antagonists on human NK cell function represent an important application of an animal to humanized IL-15. Genetically modified animals, which express human or humanized IL-15, are described which are useful for testing human IL-15 antagonists. Genetically modified animals further comprise an animal model of a human disease, for example, the disease is genetically induced (knocking or knockout) or otherwise. The non-human genetically modified animals may further comprise a compromised immune system, for example, a non-human animal genetically modified to sustain or maintain a human transplant, for example a human solid tumor or a blood cell tumor (e.g. lymphocyte tumor, e.g., a B or T cell tumor).

EXAMPLES

Example 1: Humanization of the Mouse IL-15 Locus

Mouse ES cells were modified to replace certain mouse IL-15 gene sequences with certain human IL-15 gene sequences at the locus of the endogenous mouse IL-15, under control of the mouse IL-15 regulatory elements , using VELOCIGENE® genetic engineering technology to produce a humanized locus, as shown in FIG. FIG. 1 does not show upstream (in relation to the sense of transcription of the IL-15 gene) the 5 'untranslated exons of the mouse gene; The Exl of FIG. 1 shows a small untranslated (unfilled) region upstream of the coding electron. As shown, the humanization at the bottom of FIG. 1, exons coding for mouse 1 and 2 were maintained, whereas the 3 to 6 coding exons were replaced by 3 to 6 human exons. At the downstream end, the human exon 6 is followed by a stop codon and a human 3'-UTR and further by a human sequence found downstream of the human 3'UTR. For selection purposes, a selection cassette (floxed for Cre removal) has been included. The humanized locus of FIG. 1 expressing a mature IL-15 protein which is fully human.

Direction of the Construct. Bacterial homologous recombination (BHR) is performed to construct a large targeting vector (LTVEC) containing human IL-15 gene sequences for targeting to the mouse IL-15 locus using standard BHR techniques (see, for example, Valenzuela et al. (2003) High-throughput engineering of the mouse genome coupled with high-resolution expression analysis, Nature Biotech 21 (6): 652-659) and BHR gap repair. Linear fragments are generated by the binding of the PCR-generated homology boxes to cloned cassettes followed by gel isolation of the binding and electroporation products in bacteria harboring the target bacterial artificial chromosome (BAC). Mouse BAC PRCI23-203P7 is used as the origin of the mouse sequence; BAC RP11-103B12 is used as the origin of the human IL-15 gene sequence. After a selection step, the correctly recombined clones are identified by PCR at new junctions and by restriction analysis. A large targeting vector (LTVEC) containing arms of homology and human IL-15 gene sequences was produced. Mouse ES cells were electroporated with LTVEC constructs, grown in selection medium and used as donor ES cells for humanized IL-15 mice comprising a locus replacement of the endogenous mouse IL-15 by the human sequence as depicted in FIG . The mouse IL-15 gene (GenelD from mouse: 103014; RefSeq transcribed: NM 008357.2; eld from set: 16168) is modified by the use of genomic coordinates for deletion GRCM38: ch 8: 82331173-82343471 (minus) ; genomic coordinates for GRCh37 substitution: ch4: 142642924-142655819 (plus ribbon). 12299 nucleotides of the mouse sequence were replaced by 128996 nucleotides of human sequence. Replacement of the mouse IL-15 sequence described above is shown graphically in FIG. 1. LTVEC comprising the humanized IL-15 gene had about 13 kb of upstream mouse targeting arm flanked upstream with Mlul site, and a mouse targeting arm of 27 kb downstream flanked downstream by an Ascl site. LTVEC was linearized with Mlul and Ascl for electroporation.

Following the construction of LTVEC, the LTVEC nucleotide sequence at the 5 'human / mouse junction and at the 3' human / mouse junction is shown in FIGs. 2-4.

After electroporation of the ES cell, a native allele loss assay (see, for example, Valenzuela et al. (2003)) is performed to detect the loss of the endogenous IL-15 sequence due to targeting.

Correctly targeted ES cells (MAID 5217) were further electroporated with a transient Cre expression vector to remove the Neo drug screening cassette. The resulting cassette deleted ES cells were designated MAID 5218.

Example 2: Humanized IL-15 mice Generation of mice with humanized IL-15.

Donor ES cell donors comprising a humanized IL-15 locus (e.g., MAID 5217 or MAID 5218) were introduced into early-stage mouse embryos by the VELOCOUSEUSE method (Poueymirou et al. (2007) in the generation mice F0 derived from gene-directed embryonic stem cells that allow immediate phenotypic analyzes, Nat Biotechnol 25: 91-99). Heterozygous mice are obtained, and to obtain homozygotes relative to humanized IL-15, the heterozygotes are cross-linked.

Example 3: Phenotyping of Humanized Mouse Mice IL-15.

Mice were wild-type 8-10 week Balb / c females (WT) or age-matched MAID 5217 females (heterozygotes for the human IL-15 gene). Alternatively, the mice were MAID 5217 or wild-type (WT) or age-matched MAID 5218 (both heterozygous for the human IL-15 gene) mice.

Injection of poly I: C in vivo.

Het Balb / c or MAID 5217 were injected with 50 in poly (C) (Invivogen; Cat # tlrl-pic) via caudal vein (IV injection). After 24 hours, the mice were sacrificed and blood was collected via cardiac puncture and the serum was isolated. Spleens were also harvested and splenocytes were prepared by spleens mechanically broken through a 70 μm mesh filter by treatment with Use ACK buffer (Invitrogen) to lyse red blood cells (RBCs). Isolated splenocytes were cultured by additional stimulation (see below). Serum was analyzed by human IL-15 using QUANTIKINETM human IL-15 kit from R & D Systems. The het WT or MAID 5218 mice were injected with 50æg of polyvinyl chloride (Invivogen; Cat # tlrl-pic) via IP. Mice were collected the next day via cardiac puncture and the serum was analyzed for human IL-15 by ELISA (QUANTIKINETM ELISA kit from R & D Systems).

Preparation of dendritic cells derived from bone marrow (BM-DC). The bone marrow was separated from the tibia of uninjected mice and RBCs lysed with ACK lysis buffer. Cells were washed with complete RPMI (with HEPES, Gentamicin, sodium pyruvate, L-glutamine and non-essential amino acids) + 10% fetal bovine serum (FBS) and counted. 2 x 10 6 cells were cultured per well in a 6-well plate with 3 ml / well of complete RPMI + 10% FBS + 50 ng / ml murine GM-CSF + 50 ng / ml murine IL-4. Cells were cultured at 37øC / 5% CO 2 and fresh GM-CSF / IL-4 was given on days 2 and 4 of the culture. At day 5 of the culture, the non-adherent BM-DCs were collected from the cultures and the respective culture medium was saved (conditioned media).

Culture of splenocytes.

Spleens were also collected from respective mice and splenocytes were prepared by spleens mechanically broken through a 70 pM mesh filter by treatment with ACK lysis buffer (Invitrogen) to lyse RBCs. The isolated splenocytes were cultured in a 48-well plate in 2x106 / ml of splenocytes in 1 ml of complete RPMI + 10% FBS. Cells were treated with 10æg / ml poly I: C; 10 pg / mL of PMA or untreated. Cells were cultured overnight and the next day supernatant was collected and concentrated using 8-fold Amicon 2-ml 3 kd (MWCO) molecular weight cutoff filters. The concentrated supernatants were analyzed by human IL-15 using the QUANTIKINE ™ kit of human IL-15 from R & D Systems.

Culture of BM-DC. 2 x 10 6 / ml BM-DCs were plated on a 24-well plate in 0.5 ml of fresh RPMI + 10% FBS and 0.5 ml of conditioned medium. Cells were treated with 25æg / ml poly I: C; 1 pg / mL LPS or untreated. All conditions were performed in duplicate. Cells were cultured as such for 36 hours, and then the supernatant was harvested. Supernatants were concentrated 7 times using 2 ml Amicon filters with 3 kd MWCO. Levels of human IL-15 in concentrated supernatants were analyzed using QUANTIKINETM human IL-15 kit from R & D Systems. RNA was isolated from the cells via the Qiagen RNAeasy ™ miniprep kit for analysis by RT-PCR of human IL-15 transcript levels. ELISA. The human IL-15 QUANTIKINETM kit from R & D systems was used to measure human IL-15 in serum and the concentrated splenocytes or BM-DC supernatants. The kit was used according to the manufacturer's instructions. Additional controls were performed to validate this kit for specificity (detects only human IL-15 rather than mouse IL-15) and to confirm that it does not react with poly I: C. Therefore, 1000 pg / ml murine IL-15 was run in the ELISA (note: the highest standard for human IL-15 is 250 pg / ml) and poly I: C alone at 25 pg / ml and 12.5 pg / mL. The kit was found to specifically react for human IL-15 (without detection of mouse IL-15) and unreacted with poly I: C. RT-PCR.

cDNA was prepared from 200 ng RNA isolated using SUPERSCRIPT ™ III first ribbon synthesis system for RT-PCR kit (Invitrogen) according to the manufacturer's instructions. The specific human IL-15 transcript was amplified using Taqman DNA polymerase with the following primers: Forward primer -15: gtaaraagtg atttgaaaaa aattgaagat (SEQ ID NO: 7); Reverse primer hIL-15: tacaaaactc tgcaaaaatt ctttaatat (SEQ ID NO: 8). The PCR reaction was performed with 40 cycles of the following: denaturation at 94 ° C for 15 seconds, annealing at 60 ° C for 30 seconds, extending at 72 ° C and then maintaining the reaction at 4 ° C. The transcript was run on 1% agarose gel using loading dye Promega 6x.

Results. Human IL-15 was observed in MAID 5217 het serum injected with poly I: C, but not in an age / sex coupled Balb / c mouse injected with poly I: C (Figure 6 and Figure 10, right panel). Likewise, human IL-15 was observed in MAID 5218 het serum injected with poly I: C, but not in a poly (1: C) injected age / gender-matched WT mouse (Figure 10, left panel). The level of IL-15 produced in MAID 5218 was comparable to that of MAID 5217 (Figure 10). MAID-5217 PMA-stimulated splenocytes secrete low levels of human IL-15 in vitro (were not observed in the splenocytes of WT mice).

In addition, BMDs derived from MAID 5217 het demonstrate secretion of IL-15 after in vitro stimulation with poly I: C (TLR3 agonist) and LPS (TLR4 agonist), as well as significant baseline levels. RT-PCR analysis demonstrated human specific IL-15 transcript only in BM-DCs from MAID 5217 het mice.

Overall, the data indicate that MAID 5217 het and MAID 5218 het express human IL-15.

Example 4: Homozygous mouse for Human IL-15

The heterozygous mice are crossed and then genotyped as described above. Mice with homozygous hIL-15 are maintained for breeding purposes.

SEQUENCE LISTING <110> Rojas, Jose F.

Lai, Ka-Man Venus Murphy, Andrew J. Humanized IL-15 Animals <130> 31013 (2200) <150> 61891013 <151> 2013-10-15 <160> 8 <170> Patentln version 3.5

<210> 1 <211> 208 <212> DNA <213> Sequence Artificial <220> <223> Synthetic Oligonucleotide <400> 1 atccatttag cctttctctg atcactaagt tggacagttg gacagtcttc ctcaaattag 60 cttagactat caaaattata ctgtattttt ggtatttcca gcgatcgctt cagttacaag 120 gctgttgaat gcacagaagc aaggataaca ctgatttttt cactggtcag aataaaaatt 180 attgattgct cttttgctta tagtattc 208 <210> 2 <211> 2029

<212> DNA <213> Sequence Artificial <220> <223> Synthetic Oligonucleotide <400> 2 aatgtaacag aatctggatg caaagaatgt gaggaactgg aggaaaaaaa tattaaagaa 60 tttttgcaga gttttgtaca tattgtccaa atgttcatca acacttcttg attgcaattg 120 attcttttta aagtgtttct gttattaaca aacatcactc tgctgcttag acataacaaa 180 acactcggca tttcaaatgt gctgtcaaaa caagtttttc tgtcaagaag atgatcagac 240 cttggatcag atgaactctt agaaatgaag gcagaaaaat gtcattgagt aatatagtga 300 ctatgaactt ctctcagact tactttactc atttttttaa tttattattg aaattgtaca 360 tatttgtgga ataatgtaaa atgttgaata aaaatatgta caagtgttgt tttttaagtt 420 gcactgatat tttacctctt attgcaaaat agcatttgtt taagggtgat agtcaaatta 480 tgtattggtg gggctgggta ccaatgctgc aggtcaacag ctatgctggt aggctcctgc 540 cagtgtggaa ccactgacta ctggctctca ttgacttcct tactaagcat agcaaacaga 600 ggaagaattt gttatcagta agaaaaagaa gaactatatg tgaatcctct tctttatact 660 gtaatttagt tattgatgta taaagcaact gttatgaaat aaagaaattg caataactgg 720 catataatgt ccatcagtaa atcttggtgg tggtggcaat aataaacttc tactgatagg 780 tagaatggt g tgcaagcttg tccaatcacg gattgcaggc cacatgcggc ccaggacaac 840 tttgaatgtg gcccaacaca aattcataaa ctttcataca tctcgttttt agctcatcag 900 ctatcattag cggtagtgta tttaaagtgt ggcccaagac aattcttctt attccaatgt 960 ggcccaggga aabcaaaaga ttggatgccc ctggtataga aaactaatag tgacagtgtt 1020 catatttcat gctttcccaa atacaggtat tttattttca cattcttttt gccatgttta 1080 tataataata aagaaaaacc ctgttgattt gttggagcca ttgttatctg acagaaaata 1140 attgtttata ttttttgcac tacactgtct aaaattagca agctctcttc taatggaact 1200 gtaagaaaga tgaaatattt ttgttttatt ataaatttat ttcaccttaa ttctggtaat 1260 actcactgag tgactgtggg gtgggaaatg atctcttaag aatttgattt ctttctattc 1320 catagtacaa actcgttctc tgttgaaaca ttcttctatc accccagtgc cctatccatg 1380 tacatgtgtt cttattgctc tagtcaaacg gtgcttataa atatctttca gaaagtttag 1440 gagaaatctg tatcctattt gacttccaat aatcatgtat tggctgtcag cttcttacct 1500 actctcagtc cagagaaata gtatttggca gccactcttt aaagtttatg ggttgtggat 1560 tgtggcggtt gatttatttt ttttatttca attgggatag aattttttaa tatacctgta 1620 tttttgtttt gttttat gta gcttttctat tagggagagt aggaaaagtg caccattttc 1680 ttctctaaat ttccagtcca gtctttaggg gaatgttagt cttcctgaga tgggggaagg 1740 aaaatcataa tgccagtcac tttgcaaata atattttata gtgataaatg gttcattttg 1800 gttacatagg catacaagtg ggcttaaaac ttggaattta ccagggctca aaattaaaat 1860 tcttacatta gttactcgat atggatcgct tcagttgatc ttagaaaact caaggcatag 1920 atctgcaacc tcgagataac ttcgtataat gtatgctata cgaagttata tgcatggcct 1980 ccgcgccggg ttttggcgcc tcccgcgggc gcccccctcc tcacggcga 2029 <210> 3

<211> 200 <212> DNA <213> Sequence Artificial <220> <223> Synthetic Oligonucleotide <400> 3 cattctcagt attgttttgc caagttctaa ttccatcaga cctcgacctg cagcccctag 60 ataacttcgt ataatgtatg ctatacgaag ttatgctagc gtgatagtcc ttcacggaaa 120 gtacaagaat acacagaaaa ctgctgttta cattagtctt tcacgttttt attttattct 180 cacaaatttt aatgcaatac 200 < 210> 4

<211> 162 <212> PRT <213> Artificial Sequence <22 0> <223> murine IL-15 precursor polypeptide <400> 4

Met Lye Ile Leu Lys Pro Tyr Met Arg Asn Thr Ser Ile Ser Cys Tyr 15 10 15

Leu Cys Phe Leu Leu Asn Ser His Phe Leu Thr Glu Ala Gly lie His 20 25 30

Val Phe Ile Leu Gly Cys Val Ser Val Gly Leu Pro Lys Thr Glu Ala 35 40 45

Asn Trp Ile Asp Val Arg Tyr Asp Leu Glu Lys Ile Glu Ser Leu Ile 50 55 60

Gin Ser He His lie Asp Thr Thr Leu Tyr Thr Asp Ser Asp Phe His 65 70 75 80

Pro Ser Cys Lys Val Thr Ala Met Asn Cys Phe Leu Leu Glu Leu Gin 85 90 95

Val Ile Leu His Glu Tyr Ser Asn Met Thr Leu Asn Glu Thr Val Arg 100 105 110

Asn Val Leu Tyr Leu Wing Asn Ser Thr Leu Ser Ser Asn Lys Asn Val 115 120 125

Ala Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Thr Phe 130 135 140

Thr Glu Phe Leu Gin Ser Phe Ile Arg He Val Gin Met Phe Ile Asn 145 150 155 160

Thr Ser <210> 5

<211> 162 <212> PRT <213> Artificial Sequence <220> <223> Hybrid mouse / human IL-15 precursor polypeptide <400> 5

Met Lys Ile Leu Lys Pro Tyr Met Arg Asn Thr Ser Ile Ser Cys Tyr 15 10 15

Leu Cys Phe Leu Leu Asn Ser His Phe Leu Thr Glu Ala Gly lie His 20 25 30

Val Phe Ile Leu Gly Cys Phe Ser Ala Gly Leu Pro Lys Thr Glu Ala 35 40 45

Asn Trp Val Asn Valine Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile 50 55 60

Gin Ser Met His Lie Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His 65 70 75 80

Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gin 85 90 95

Val Xle Ser Leu Glu Ser Gly Asp Ala Ser lie His Asp Thr Val Glu 100 105 110

Asn Leu lie Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val 115 120 125

Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn lie 130 135 140

Lys Glu Phe Leu Gin Ser Phe Val His lie Val Gin Met Phe lie Asn 145 150 155 160

Thr Ser

<210> 6 <211> 162 <212> PRT <213> Artificial Sequence <220> <223> human IL-15 isoform 1 polypeptide <400> 6

Met Arg Ile Ser Lys Pro His Leu Arg Ser Ile Ser Ile Gin Cys Tyr 15 10 15

Leu Cys Leu Leu Leu Asn Ser His Phe Leu Thr Glu Ala Gly lie His 20 25 30

Val Phe Ile Leu Gly Cys Phe Ser Ala Gly Leu Pro Lys Thr Glu Ala 35 40 45

Asn Trp Val Asn Valine Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile 50 55 60

Gin Ser Met His Lie Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His 65 70 75 80

Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gin 85 90 95

Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu 100 105 110

Asn Leu lie Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val 115 120 125

Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn He 130 135 140

Lys Glu Phe Leu Gin Ser Phe Val His He Val Gin Met Phe He Asn 145 150 155 160

Thr Ser <210> 7 <211> 30

<212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide: hIL-15 Forward primer <400> 7 gtaaraagtg atttgaaaaa aattgaagat 30 <210> 8 <211> 29

<212> DNA <213> Artificial Sequence <22 0> <223> Synthetic Oligonucleotide: hIL-15 Reverse primer <400> 8 tacaaaactc tgcaaaaatt ctttaatat 29

Claims (12)

A genetically modified mouse characterized in that the genome comprises a substitution at a mouse endogenous IL-15 locus of a mouse genomic fragment encoding a mature mouse IL-15 polypeptide by a human genomic segment which encodes a human mature IL-15 polypeptide, wherein the human IL-15 exons in said human genomic segment consist of exons 3, 4, 5 and 6 encoding the human IL-15 protein to form an IL-15 gene , wherein the exons of IL-15 are numbered with the first exon coding as exon 1, and wherein the humanized IL-15 gene is under the control of all regulatory elements upstream of the endogenous mouse IL-15 in the mouse locus of mouse endogenous IL-15. A genetically modified mouse according to claim 1, wherein the gene encoding humanized IL-15 encodes a protein comprising the amino acid sequence of SEQ ID NO: 5. A genetically modified mouse according to claim 1, wherein the human genomic segment further comprises a human intron sequence upstream of the human exon, nearest to 5 ', and a human protein non-coding sequence downstream of the codon and downstream of human 3 'UTR. A method of producing a genetically engineered mouse comprising modifying the genome of a mouse by replacing a mouse genomic fragment encoding a mature mouse IL-15 polypeptide at the IL- 15 protein by a human genomic segment encoding a mature human IL-15 polypeptide, wherein the human IL-15 exons in said human genomic segment consist of exons 3, 4, 5 and 6 encoding the IL- 15 to form a humanized IL-15 gene, wherein the exons of IL-15 are numbered with the first exon coding as exon 1, and wherein the humanized IL-15 gene is under the control of all regulatory elements upstream of the endogenous mouse IL-15 at the mouse endogenous IL-15 locus. A method according to claim 4, wherein the human genomic segment further comprises a human intron sequence upstream of the human exon, nearest to 5 ', and a human protein non-coding sequence downstream of the stop codon human and downstream of human 3 'UTR. A method according to claim 4, wherein the gene encoding the humanized IL-15 encodes a protein comprising the amino acid sequence of SEQ ID NO: 5. A genetically modified mouse whose genome comprises a humanized IL-15 gene, wherein said humanized IL-15 gene comprises exons 1 and 2 encoding the mouse IL-15 protein and a human genomic segment, wherein Exons of human IL-15 in said human genomic segment consist of exons 3, 4, 5 and 6 encoding the human IL-15 protein, wherein the IL-15 exons are numbered with the first exon coding as exon 1, and in that the humanized IL-15 gene is under the control of all regulatory elements upstream of the mouse IL-15 from a mouse endogenous IL-15 locus, and said humanized IL-15 gene encodes a protein comprising a mature human IL-15 polypeptide sequence. A genetically modified mouse according to claim 7, wherein the gene encoding humanized IL-15 encodes a protein comprising the amino acid sequence of SEQ ID NO: 5. A genetically modified mouse according to claim 7, wherein the human genomic segment further comprises a human intron sequence upstream of the human exon, nearest to 5 ', and a human protein non-coding sequence downstream of the codon and downstream of human 3 'UTR. A genetically modified mouse according to claim 7, wherein the humanized IL-15 gene is at a position in the mouse genome which is different from the endogenous IL-15 locus. A method of producing a genetically modified mouse comprising the introduction into the genome of a mouse of a humanized IL-15 gene comprising exons 1 and 2 encoding the mouse IL-15 protein and a human genomic segment, wherein the exons of human IL-15 in said human genomic segment consist of exons 3, 4, 5 and 6 of human IL-15, wherein the exons of IL-15 are numbered with the first coding exon as exon 1, and wherein the humanized IL-15 gene is under the control of all regulatory elements upstream of the mouse IL-15 of a locus of the endogenous mouse IL-15, and said IL-15 gene encodes a protein comprising a mature human IL-15 polypeptide sequence. A method according to claim 11, characterized in that the humanized IL-15 gene is integrated into the genome at a position which is different from the endogenous IL-15 locus.